Antibiotics X-14873 A, G and H

ABSTRACT

There are provided antibiotics X-14873 A, G and H of the formulas ##STR1## wherein for X-14873A, R 1  is CO 2  H and R 2  is ##STR2## for X-14873G, R 1  is hydrogen and R 2  is ##STR3## and for X-14873H, R 1  is hydrogen and R 2  is ##STR4## The compounds exhibit antibiotic activity. Also disclosed is a process to produce the above compounds.

DESCRIPTION OF THE INVENTION

The present invention relates to Antibiotics A, G, and H of the formula##STR5## wherein for X-14873A, R₁ is CO₂ H and R₂ is ##STR6## forX-14873G, R₁ is hydrogen and R₂ is ##STR7## and for X-14873H, R₁ ishydrogen and R₂ is ##STR8##

Also included within the scope of the present invention are thepharmaceutically acceptable salts thereof.

As utilized in the structural formulas herein the expression Me standsfor methyl and Et stand for ethyl.

There is further provided, according to the present invention, afermentation process for the production of such antibiotic substancestogether with the isolation techniques utilized to recover theantibiotic compound from the fermentation broth. In addition, theisolation of three novel actinomycins, X-14873 B, C and D, is described.

The organism producing the antibiotics of the present invention is a newspecies designated Streptomyces sp. X-14873. A culture of the livingorganism, given the laboratory designation X-14873 has been deposited inthe American Type Culture Collection, Rockville, Md. and added to itspermanent collection of microorganisms as ATCC 31679. The newmicroorganism was isolated from a soil sample near sagebrush in CrandallCreek, Wyoming.

Growth Characteristics

The organism was cultivated on the standard ISP media (Difco) describedby Shirling and Gottlieb "Methods for Characterization of StreptomycesSpecies", Intern. J. System. Bacteriol., 16, pp 313-340, 1966

Media utilized in other tests were those from the following references:

    __________________________________________________________________________    Test                  References                                              __________________________________________________________________________    A.                                                                              Sodium Chloride tolerance                                                                         Gordon and Smith "Rapidly                               B.                                                                              Hydrolysis of casein                                                                              Growing Acid Fast                                       C.                                                                              Reduction of nitrate                                                                              Bacteria", J. Bacteriol.,                                                     66:41-48, 1953                                          D.                                                                              Gelatin (modified with nutrient gelatin                                                           Skerman, "A Guide to the                                  (BBL) plus 2.00% agar in place of meat                                                            Identification of the Genera                              infusion agar)      of Bacteria", Williams and                                                    Williams Co., Baltimore, 1967.                          E.                                                                              Starch (Actinomyces broth [Difco]                                             plus 0.25% soluble starch and 2.0% agar)                                    F.                                                                              Decomposition of adenine, xanthine,                                                               Gordon, "The taxonomy of                                  tyrosine and hypoxanthine                                                                         soil bacteria", p. 293-321,                                                   Gray and Parkinson, Ecology                                                   of Soil Bacteria, Liverpool                                                   Press, Liverpool, England.                              __________________________________________________________________________

Tests were run at 28° and 37° C. for almost all media. Colordeterminations were made after 2 and 4 weeks of incubation. Pigmentationwas described by using the color scheme in the Color Harmony Manual, 4thed., 1958.

Carbon utilization was determined by the method of Shirling and Gottlieb(above) using ISP-9 (Difco) medium.

A 48 hour-old ISP-1 broth culture of X-14873 was centrifuged andhomogenized to obtain a washed suspension for inoculation.

The ability of the organisms to grow at 10°, 28°, 37°, 45° and 50° C.was investigated by inoculating broth of ISP-1 (Difco) medium. Cell wallanalysis of the isomer of diaminopimelic acid was performed by themethod of Becker et al., Appl. Microbiol., 12, 421-423, 1964.

Results

Microscopic Examination

Strain X-14873 produces a substrate mycelium which does not fragmentinto spores and an aerial mycelium, forming rectus-flexibilis sporechains with 10-20 spores per chain. Spores are smooth and range in sizefrom 1.0×0.52 μm to 1.25 to 0.75 μm.

Cell Wall Analysis

The cell wall contains the LL-isomer of diaminopimelic acid which,together with the above microscopic examination, places this organism inthe genus Streptomyces.

Macroscopic Examination

Table 1 summarizes the amount of growth, degree of sporulation, sporemass color, color of reverse-substrate mycelium and presence of solublepigment produced by strain X-14873 on various agar media.

                                      TABLE 1                                     __________________________________________________________________________    Cultural characteristics of strain X-14873                                    Agar     Amount of Growth    Color of Reverse                                 Medium   Degree of Sporulation                                                                    Spore Mass Color                                                                       Substrate Mycellium                              __________________________________________________________________________    Yeast-malt                                                                             moderate to abundant                                                                     c(light gray)                                                                          3ie(camel) and                                   extract (ISP2)                                                                         growth; moderate                                                                         where sporu-                                                                           2gc(light tan) at                                         sporulation; brown                                                                       lated; 3ni(clove                                                                       edge                                                      soluble pigment                                                                          brown) where not                                                              sporulated                                                Oatmeal (ISP3)                                                                         moderate growth;                                                                         b(oyster white)                                                                        2ec(biscuit)                                              sparse sporulation;                                                                      where sporu-                                                       yellow soluble                                                                           lated; 2gc                                                         pigment    (bamboo) where                                                                not sporulated                                            Inorganic salts-                                                                       abundant growth; well                                                                    3cb(sand)                                                                              3lc(amber) and                                   starch (ISP4)                                                                          sporulated; hydrolyzes                                                                            3le(cinnamon)                                             starch; yellow soluble                                                        pigment                                                              Glycerol-                                                                              moderate growth;                                                                         b(oyster white)                                                                        3le(cinnamon)                                    Asparagine (ISP5)                                                                      moderate sporulation;                                                         yellow-brown soluble                                                          pigment; hygroscopic                                                 Czapek-Dox                                                                             moderate growth;                                                                         b(oyster white)                                                                        2ic(honey gold)                                           moderate sporulation;                                                         yellow soluble                                                                pigment; slightly                                                             hygroscopic                                                          __________________________________________________________________________

Physiological Characteristics

Strain X-14873 hydrolyzes casein, starch, gelatin, adenine, xanthine,hypoxanthine, tyrosine and urea. Table 2 compares the carbon utilizationcharacteristics of strain X-14873 with those of Streptomyceschrysomallus, Streptomyces parvus and Streptomyces globisporus chosenfor their similarity in morphological and physiological characteristics.

                                      TABLE 2                                     __________________________________________________________________________    Comparison of carbon utilization by strain X-14873                            and related strains                                                                     Result.sup.a                                                                       S. chrysomallus                                                                       S. parvus                                                                             S. globisporus                                 Carbon source                                                                           X-14873                                                                            3194A   3195A   3201A                                          __________________________________________________________________________    No carbon control                                                                       -    -       -       -                                              D-Glucose ++   ++ YS   ++ YS   ++ YS                                          D-Xylose  ± +       ++ YS   +                                              L-Arabinose                                                                             +    +       +(+)    +                                              L-Rhamnose                                                                              ++ YS                                                                              ++ YS   ++      ++                                             D-Fructose                                                                              ++   ++      ++      ++                                             D-Galactose                                                                             ++   ++ YS   ++ YS   ++                                             Raffinose ± -       -       -                                              D-Mannitol                                                                              ++ YS                                                                              ++ YS   ++ YS   ++                                             i-Inositol                                                                              ± -       -       -                                              Salicin   +    ±    ±    ±                                           Sucrose   -    -       -       -                                              Cellulose -    -       -       -                                              Maltose   -    ++ YS   ++ intense YS                                                                         ++                                             Glycerol  ++   ++      ++ intense YS                                                                         ++                                             Starch    ++   ++ YS   ++      ++                                             Ribose    ++   ++ YS   ++ YS   ++ YS                                          __________________________________________________________________________     .sup.a -: Negative response; ±: doubtful response; +: more growth than     on carbon control but less than on glucose; +(+): positive response,          nearly equal to growth on glucose; ++: positive response equal to growth      on glucose. YS = yellow soluble pigment.                                 

Further metabolic and morphological characteristics are set forth inTable 3 below.

                  TABLE 3                                                         ______________________________________                                        Test             Result                                                       ______________________________________                                        Spore mass color gray(yellowish)                                              ISP6, darkening  -                                                            Melanin, ISP7    -                                                            ISP1, darkening  -                                                            Casein hydrolysis                                                                              +                                                            Starch hydrolysis                                                                              +                                                            NaCl(%) tolerance                                                                              5                                                            Growth range temp(°C.)                                                                  10-28°                                                Reverse-side pigment                                                                           -                                                            Soluble pigment  yellow                                                       Streptomycin sensitivity                                                                       +13mm                                                        (10 mcg in 1/4" disc)                                                         Lysozyme sensitivity                                                                           +                                                            Nitrate reduction                                                                              +                                                            Hygroscopic property                                                                           +                                                            Antibiotic Production                                                                          Antibiotic X-14873A                                                           Antibiotic X-14873G                                                           Antibiotic X-14873H                                                           Actinomycin X-14873B                                                          Actinomycin X-14873C                                                          Actinomycin X-14873D                                         ______________________________________                                    

Discussion and Conclusion

Streptomyces strain X-14873 resembles S. chrysomallus, S. parvus, and S.globisporus. All but S. globisporus produce actinomycin. The three knowncultures were also tested for production of hydroxylysocellin. In abioautogram vs. Bacillus sp. (ATCC 27860), both S. chrysomallus and S.parvus produced activity at a similar R_(F) in the two different solventsystems.

The known cultures possess a yellow-colored spore mass, while X-14873 ismainly gray with only a hint of yellow. Strain X-14873 produces a yellowsoluble pigment as do the other strains. The carbon utilization patternsare very similar for all the strains, except X-14873 does not utilizemaltose. Since the cultures all hydrolyze starch, gelatin, casein, andurea, and decompose adenine, xanthine, hypoxanthine, and tryosine, itwould be difficult to distinguish between all these cultures.

Strain X-14873 cannot be assigned to a particular species, but insteadresembles a group which could be called the Streptomyces chrysomallus-S.parvus group based on similarity in antibiotic, yellow soluble pigmentproduction, and carbon utilization pattern.

The strain Streptomyces X-14873 described herein includes all strains ofStreptomyces which form the compounds X-14873A, B, C, D, G and H andwhich cannot be definitely differentiated from the culture numberX-14873 and its subcultures, including mutants and variants.

Streptomyces sp. X-14873 when grown under suitable conditions, producesa compound of Formula I. A fermentation broth containing StreptomycesX-14873 is prepared by inoculating spores or mycelia of the organismproducing the compound of Formula I into a suitable medium and thencultivating under aerobic conditions. For the production of a compoundof the Formula I, cultivation on a solid medium is possible, but forproduction in large quantities, cultivation in a liquid medium ispreferable. The temperature of the cultivation may be varied over a widerange, 20°-35° C., within which the organism may grow, but a temperatureof 26°-30° C. and a substantially neutral pH are preferred. In thesubmerged aerobic fermentation of the organism for the production of acompound of the Formula I, the medium may contain as the source forcarbon a commercially available glyceride oil or a carbohydrate such asglycerol, glucose, maltose, lactose, dextrin, starch, etc. in pure orcrude states and as the source of nitrogen, an organic material such assoybean meal, distillers' solubles, peanut meal, cotton seed meal, meatextract, peptone, fish meal, yeast extract, corn steep liquor, etc. andwhen desired, inorganic sources of nitrogen such as nitrates andammonium salts and mineral salts such as ammonium sulfate, magnesiumsulfate and the like. It also may contain sodium chloride, potassiumchloride, potassium phosphate and the like and buffering agents such assodium citrate, calcium carbonate or phosphates and trace amounts ofheavy metal salts. In aerated submerged culturing procedures, ananti-foam agent such as liquid paraffin, fatty oils or siliconecompounds is used. More than one kind of carbon source, nitrogen sourceor anti-foam source may be used for production of a compound of theFormula I.

Considered within the ambit of the present invention are the organic orinorganic pharmaceutically acceptable salts of the compound of FormulaI. These salts are prepared from the free acid by methods well known inthe art--for example, by washing the free acid in solution with asuitable base or salt. Examples of such pharmaceutically acceptablebasic substances capable of forming salts for the purpose of the presentinvention include alkali metal bases such as sodium hydroxide, potassiumhydroxide, lithium hydroxide and the like alkaline earth metal basessuch as calcium hydroxide, barium hydroxide and the like, and ammoniumhydroxide. Alkali metal or alkaline earth metal salts suitable forforming pharmaceutically acceptable salts can include anions such ascarbonate, bicarbonate and sulfates. Preferred for use in this inventionare salts formed from alkali metal bases.

Examples of organic bases forming pharmaceutically acceptable salts withthe compound of Formula I are lower alkyl amines, primary, secondary andtertiary hydroxy-lower alkylamines such as ethylamine, isopropylamine,diethylamine, methyl-n-butylamine, ethanolamine and diethanolamine.

The following examples serve to illustrate this invention withoutlimiting it thereto:

EXAMPLE 1 Shake-flask Fermentation

The Streptomyces X-14873 culture is grown and maintained on astarch-casein agar slant having the following composition (grams/literdistilled water):

    ______________________________________                                        Soluble starch    10.0                                                        Casein            1.0                                                         K.sub.2 HPO.sub.4 0.5                                                         MgSO.sub.4 (anhydrous)                                                                          0.5                                                         Agar              20.0                                                        The medium pH is adjusted to 7.4 with NaOH before autoclaving.                ______________________________________                                    

The slant is inoculated with X-14873 culture and incubated at 28° C. for7-14 days. A chunk of agar containing spores and mycelia from thesporulated culture is used to prepare vegetative inoculum by inoculatinga 500-ml Erlenmeyer flask containing 100 ml of inoculum medium havingthe following composition (grams/liter distilled water):

    ______________________________________                                        Tomato pomace    5.0                                                          Distillers soluble                                                                             5.0                                                          OM peptone       5.0                                                          Debittered yeast 5.0                                                          Corn starch      20.0                                                         CaCO.sub.3       1.0                                                          K.sub.2 HPO.sub.4                                                                              1.0                                                          pH is adjusted to 7.0 before sterilization.                                   ______________________________________                                    

The inoculated inoculum medium is incubated at 28° C. for 72 hours on arotary shaker, operating at 250 rpm with a 2-inch stroke.

A 30 ml portion of the resulting culture is then used to inoculate a6-liter Erlenmeyer flask containing 1.25 liter sterilized productionmedium having the following composition (grams/liter distilled water):

    ______________________________________                                        Tomato pomace    5.0                                                          Distillers soluble                                                                             5.0                                                          OM peptone       5.0                                                          Debittered yeast 5.0                                                          Corn starch      20.0                                                         CaCO.sub.3       1.0                                                          K.sub.2 HPO.sub.4                                                                              1.0                                                          pH is adjusted to 7.0 before sterilization.                                   ______________________________________                                    

The inoculated medium is incubated at 28° C. for 5 days on a rotaryshaker running at 250 rpm with a 2-inch stroke.

EXAMPLE 2 Tank Fermentation

The Streptomyces X-14873 culture is grown and maintained on astarch-casein agar slant having the following composition (grams/literdistilled water):

    ______________________________________                                               Soluble starch                                                                          10.0                                                                Casein    1.0                                                                 K.sub.2 HPO.sub.4                                                                       0.5                                                                 MgSO.sub.4                                                                              0.5                                                                 Agar      20.0                                                         pH is adjusted to 7.4 with NaOH before autoclaving.                           ______________________________________                                    

The slant is inoculated with X-14873 culture and incubated at 28° C. for7-14 days. A chunk of agar containing spores and mycelia from thesporulated culture is used to prepare vegetative inoculum by inoculatinga 500-ml Erlenmeyer flask containing 100 ml of inoculum medium havingthe following composition (grams/liter distilled water):

    ______________________________________                                        Tomato pomace    5.0                                                          Distillers soluble                                                                             5.0                                                          OM peptone       5.0                                                          Debittered yeast 5.0                                                          Corn starch      20.0                                                         CaCO.sub.3       1.0                                                          K.sub.2 HPO.sub.4                                                                              1.0                                                          pH is adjusted to 7.0 with NaOH before sterilization.                         ______________________________________                                    

The inoculated medium is incubated for 72 hours at 28° C. on a rotaryshaker operating at 250 rpm, 2-inch stroke.

Twenty ml (1%, v/v) of this culture are used to inoculate a 6-literErlenmeyer flask containing 2 liters of medium having the followingcomposition (grams/liter distilled water):

    ______________________________________                                        Tomato pomace    5.0                                                          Distillers soluble                                                                             5.0                                                          OM peptone       5.0                                                          Debittered yeast 5.0                                                          Corn starch      20.0                                                         CaCO.sub.3       1.0                                                          K.sub.2 HPO.sub.4                                                                              1.0                                                          pH is adjusted to 7.0 before autoclaving at 15-20 pound pressure              for 45 minutes.                                                               ______________________________________                                    

The inoculated medium is incubated for 72 hours at 28° C. on a rotaryshaker operating at 250 rpm.

Four liters of this culture are used to inoculate 60 gallons of thefollowing medium in a 100 gallon fermentor (grams/liter tap water):

    ______________________________________                                        Tomato pomace     5.0                                                         Distillers soluble                                                                              5.0                                                         OM peptone        5.0                                                         Debittered yeast  5.0                                                         Corn starch       20.0                                                        CaCO.sub.3        1.0                                                         K.sub.2 HPO.sub.4 1.0                                                         SAG 4130 Antifoam 0.1                                                         (Union Carbide)                                                               The pH of the medium is adjusted to 7.0 with NaOH before                      sterilization for 11/4 hours with 60 lb/in.sup.2 steam.                       ______________________________________                                    

The inoculated medium is aerated with compressed air at a rate of 3cubic feet per minute and is stirred with agitators at 280 rpm. Thefermentation is carried out at 28° C. for 5 days.

EXAMPLE 3 Isolation of Antibiotic X-14873-Na salt from Shake FlaskFermentation of Example 1

Step A

The whole broth from four six-liter Erlenmeyer flasks each containing1.25 liters, after 5 days of fermentation was extracted twice with equalvolume of ethyl acetate. After stirring for one half hour, solvent layerwas separated and concentrated to an oil (3 g) under reduced pressure.The oil was dissolved in methylene chloride and was chromatographed on amethylene chloride slurry packed 150 g silica gel (Davison grade 62)column. The column was eluted with a gradient between 4 liters ofmethylene chloride to 4 liters of hexane/acetone (7/3) and then 3 litersof methylene chloride/methyl alcohol (8/2). Fractions of forty ml eachwere collected and from fraction numbers 181-480 were pooled, solventwas removed under reduced pressure, and the (1.20 g) residue thusobtained was dissolved in ethyl acetate and was chromatographed on anethyl acetate slurry packed 50 g silica gel (Davison grade 62) column.The column was eluted with a gradient between 4 liters ethylacetate/hexane (7/3) to 4 liters ethyl acetate and then 2 litersmethylene chloride/methyl alcohol (8/2). Fractions of forty ml each werecollected and from fraction number 41-160 were pooled, solvent wasremoved under reduced pressure, and the residue thus obtained wasdissolved in ethyl acetate and was washed with equal volume of 1 N HCltwo times, followed by washing with equal volume of Na₂ CO₃ (saturatedat room temperature) two times. The solvent phase was dried over Na₂SO₄. Solvent was removed under reduced pressure and crystallization fromacetonitrile yielded antibiotic X-14873A-Na salt Mp. 154° C.

Microanalysis: calcd for C₃₅ H₆₁ O₁₁ Na (680.87): calcd: %C, 61.73; %H,9.05; %Na, 3.38; found: %C, 61.90; %H, 9.01; %Na, 3.11; [α]_(D) ²⁵ -4.5°(CHCl₃,1%), -2.6° (CH₃ OH, 1%).

EXAMPLE 4 Isolation of Antibiotic X-14873A-Na Salt and actinomycinsX-14873B, X-14873C and X-14387D from Tank Fermentation of Example 2

Step A

To the whole broth from two sixty gallons (475 liters) fermentation wasadded, after 118 hrs. growth, consecutively twice one half volume ofmethylene chloride. After stirring for one hour, the solvent layer wasseparated. Two half volumes of methylene chloride extracts were pooledand were washed twice with one half volume of 1 N HCl, followed bywashing with one half volume of 1 N NaOH, followed by washing with equalvolume of deionized water. The solvent phase was then concentrated to anoil under reduced pressure. The 336 g oil was dissolved in n-hexane andwas extracted once with acetonitrile. A precipitate was obtained. Thiswas dissolved in water and was extracted with equal volume of methylenechloride. This methylene chloride phase was evaporated to dryness inreduced pressure and the resulting 35 g solids were dissolved in 200 mlof acetone/acetonitrile (60/40) and was chromatographed in the WatersPrep LC™/System 500 using two 370 g Prep PAK-500/C₁₈ column withacetonitrile/water (80/20) elution. Fractions of 200 ml each werecollected and from fraction numbers 3-5 and 10-14 were pooled.

Step B

The acetonitrile extract of Example 4/Step A was evaporated underreduced pressure and 18 g of the 89 g residue was dissolved in 150 ml ofacetonitrile and was chromatographed on the Waters Prep LC™/System 500using one Prep PAK-500/C₁₈ column, with acetonitrile/water (9/1)elution. Fractions of 200 ml each were collected and from fractionnumbers 1-3 and 4-7 were pooled. 71 g of the 89 g residue of theacetonitrile extract was dissolved in methyl alcohol and waschromatographed on a Sephadex LH-20 gel column with methyl alcohol.Fractions of 40 ml each were collected and fraction numbers 60-200 werepooled, solvent was removed under reduced pressure, and the 45 g residuethus obtained was dissolved in acetone/acetonitrile (60/40) and waschromatographed on the Waters Prep LC™/System 500 using two 370 g PrepPAK-500/C₁₈ column with acetonitrile/water (80/20). Fractions of 100 mleach were collected, and fraction numbers 2-4, 5-8, 9-11 and 12-18 werepooled.

Step C

Fractions 10-14 of Example 4/Step A, fraction B 4-7 and 12-18 of Example4/Step B were pooled. The solvent was removed in reduced pressure andthe 19.3 g residue thus obtained was dissolved in methylene chloride andwas subjected to chromatography on a methylene chloride slurry packed600 g silica gel (Davison grade 62) column. The column was eluted with 3liters of methylene chloride and then a gradient between 6 liters ofmethylene chloride to 6 liters of methylene chloride/methyl alcohol(95/5). Fractions of forty ml each were collected and fraction numbers289-360 were pooled. The solvent was removed under reduced pressure andcrystallization from acetonitrile yielded antibiotic X-14873A-Na salt.Mp. 152° C.

Microanalysis: Calcd for C₃₅ H₆₁ O₁₁ Na (680.87): calcd: %C, 61.73; %H,9.05; %Na, 3.38; found: %C, 61.08; %H, 9.24; %Na, 3.15; [α]_(D) ²⁵ -5.4°(CHCl₃, 1%).

Step D

Fractions 3-5 of Example 4/Step A, fractions 1-3 of Example 4/Step B,and fractions 2-4 of Example 4/Step B (of the two HPLC run) were pooled.Solvent was removed under reduced pressure and the 21 g residue thusobtained was triturated with diethyl ether. 14 g diethyl ether insolublesolids were filtered off and were chromatographed on a methylenechloride slurry packed silica gel (Davison grade 62) column. The columnwas eluted with a gradient between 6 liters methylene chloride and 8liters methylene chloride/ethyl alcohol/hexane (95/5/10) and then with 2liters methylene chloride/ethyl alcohol (95/5) and 2 literschloroform/acetone/methyl alcohol (1/1/0.1). Fractions of forty ml eachwere collected. Fraction numbers 342-410; 423-466 and 620-646 werepooled. Each pool was concentrated in reduced pressure, and by theaddition of diethyl ether, orange precipitates of actinomycin X-14873-B(1.4 g), actinomycin X-14873-C (1.2 g) and actinomycin X-14873-D (3.9 g)were obtained. Actinomycin X-14873-B, -C and -D are distinguishable onsilica gel TLC plate using methylene chloride/ethyl alcohol/n-hexane(9/1/1).

Microanalysis:

Actinomycin X-14873-B; Found %C, 57.42, 57.37; %H, 6.98, 7.00; %N,12.75, 12.67; [α]_(D) ²⁵ -244.5° (MeOH, 1%) -349.6° (CHCl₃, 1%)

Actinomycin X-14873-C: Found %C, 58.12, 57.97; %H, 7.06, 7.01; %N,13.00, 12.95; [α]_(D) ²⁵ -299.1° (MeOH, 1%) -395.2° (CHCl₃, 1%)

Actinomycin X-14873-D: Found %C, 56.50, 56.49; %H, 6.80. 7.00; %N,12.06, 12.16; [α]_(D) ²⁵ +11° (MeOH, 0.2%), -145.5° (CHCl₃, 1%)

Mass spectral analysis by fast atom bombardment of actinomycins X-14873Cand X-14873D has confirmed that the two antibiotics are isomeric, with amolecular formula of C₆₁ H₈₆ N₁₂ O₁₈ (m.w. 1275.44), calcd %C, 57.44;%H, 6.80; %N, 13.18. These results confirm the novelty of X-14873C and Dwhich, like X-14873B, give on hydrolysis inter alia, both proline and3-hydroxy-5-methylproline, clearly distinguishing all three from anyknown actinomycin.

Activities of the three actinomycins are:

    ______________________________________                                                              Antitumor vs.                                                        Acute    S-180 tumor implant                                                  Mouse LD.sub.50                                                                        In Mice (mg/kg)                                                      (mg/kg)  active                                                  Antibiotic     i.p.   p.o.    level  toxic level                              ______________________________________                                        Actinomycin X-14873                                                                        B     0.16   2.9   0.048  0.096                                             C    >1000              6.25                                                  D   71>1000    6-12     25                                         ______________________________________                                    

In addition, actinomycin X-14873B was active in vivo as a coccidiostatin poultry. This is the first time such activity has been observed foran actinomycin.

EXAMPLE 5

The Streptomyces X-14873 culture is grown and maintained on astarch-casein agar slant having the following composition (grams/literdistilled water):

    ______________________________________                                        Soluble starch    10.0                                                        Casein            1.0                                                         K.sub.2 HPO.sub.4 0.5                                                         MgSO.sub.4 (anhydrous)                                                                          0.5                                                         Agar              20.0                                                        The medium pH is adjusted to 7.4 with NaOH before autoclaving.                ______________________________________                                    

The slant is inoculated with X-14873 culture and incubated at 28° C. for7-14 days. A chunk of agar containing spores and mycelia from thesporulated culture slant is used to prepare vegetative inoculum byinoculating a 500-ml Erlenmeyer flask containing 100 ml of inoculummedium having the following composition (grams/liter distilled water):

    ______________________________________                                        Tomato pomace    5.0                                                          Distillers soluble                                                                             5.0                                                          OM peptone       5.0                                                          Debittered yeast 5.0                                                          Eclipse N starch 20.0                                                         CaCO.sub.3       1.0                                                          K.sub.2 HPO.sub.4                                                                              1.0                                                          pH is adjusted to 7.0 before sterilization.                                   ______________________________________                                    

The inoculated medium is incubated at 28° C. for 4 days on a rotaryshaker, operating at 250 rpm with a 2-inch stroke.

A 10-ml portion of this culture is used to inoculate a 6-literErlenmeyer flask containing 2 liters of inoculum medium having the samecomposition described above.

The inoculated medium in the 2-liter Erlenmeyer flask is incubated for 3days at 28° C. on a rotary shaker operating at 250 rmp.

Six liters of the resulting culture broth are then used to inoculate a100-gallon fermentor containing 60 gallons of the same inoculum mediumdescribed above. The medium in the fermentor is supplemented with 0.1gram per liter of antifoam SAG 4130 (Union Carbide) and sterilized for11/4 hours with 60 lb/in² steam.

The inoculated fermentor is aerated with compressed air at a rate of 3cubic feet per minute and is stirred with agitators at 220 rpm. After 3days of incubation, 11 gallons of the resulting fermentation broth areused to inoculate 360 gallons of the following medium in a 500-gallonfermentor (grams/liter tap water):

    ______________________________________                                        Cerelose           20.0                                                       Peptone (Special R)                                                                              5.0                                                        (Wilson)                                                                      NaCl               5.0                                                        CaCO.sub.3         2.0                                                        Na-propionate      1.0                                                        SAG 4130 Antifoam, 0.1                                                        (Union Carbide)                                                               Adjust pH to 7.2 with NaOH before sterilization for 45 minutes                with 60 lb/in.sup.2 steam.                                                    ______________________________________                                    

The inoculated medium in the fermentor is aerated with compressed air at20 cubic feet per minute, and is stirred with agitators at 280 rpm. Thefermentation is carried out at 28° C. for 139 hours.

EXAMPLE 6 Isolation of Antibiotic X-14873G from Tank Fermentation ofStreptomyces Culture X-14873

Step A

To the whole broth of a 350 gallon (1330 liters) fermentation was addedafter 139 hrs growth an equal volume of ethyl acetate. After stirringfor one hour, the solvent layer was separated and concentrated to 5.75liters under reduced pressure, and was washed in turn with 1 N HCl,saturated Na₂ CO₃ and deionized water.

The solvent phase was then concentrated until crude antibioticX-14873A-Na crystallized out. The mother liquor was concentrated to anoil under reduced pressure. The oil was redissolved in methylenechloride and was chromatographed on a methylene chloride slurry packed 4kg silica gel (Davison grade 62) column. The column was eluted with agradient between 2 liters of methylene chloride and 6 litershexane-methylene chloride (1/1), followed by 8 liters hexane-acetone(9/1). Fractions of 500 ml each were collected, and fraction numbers 1-4and 5-8 were pooled.

Step B

The pooled fractions 1-4 of Step A were evaporated to dryness (81 g)under reduced pressure and were redissolved in methylene chloride andrechromatographed on a methylene chloride slurry packed 700 g silica gelcolumn. The column was eluted with a gradient between 3 liters methylenechloride-hexane (1/1) to 4 liters hexane-acetone (95/5), followed by 7liters hexane-acetone (9/1). Fractions of 40 ml each were collected andfraction numbers 52-310 and 320-390 were pooled. The solvent was removedunder reduced pressure and crystallization of the pool 320-390 fromdiethyl ether-hexane yielded antibiotic X-14873G. Mp. 152°-3° C.

Microanalysis calcd. for C₃₄ H₆₀ O₈ (596.82): Calcd: %C, 68.42; %H,10.13; Found: %C, 68.28; %H, 10.10; [α]_(D) ²⁵ +5.5° (CHCl₃, 1%).

Step C

The pooled fractions 5-8 of Step A were evaporated to dryness underreduced pressure, and the residue (80 g) was redissolved in methylenechloride and was rechromatographed on a methylene chloride slurry packed1 kg silica gel column. The column was eluted with a gradient between 4liters methylene chloride and 8 liters hexane-acetone (9/1), followed by6 liters hexane-acetone (8/2), 3 liters hexane-acetone (6/4) and 3liters methylene chlorideacetone (8/2). Fractions of 40 ml each werecollected. Fraction numbers 115-160, 161-182 and 183-270 were pooled,and solvent was removed under reduced pressure. Crystallization frommethylene chloride-hexane of the residue of pool 115-160 yieldedadditional antibiotic X-14873G. Crystallization from methylenechloride-hexane of the residue of pool 183-270 yielded antibioticX-14873H. Mp. 145°-6° C.

Microanalysis calcd for C₃₄ H₆₂ O₉ (614.84): Calcd: %C 66.41; %H, 10.16;Found:

    ______________________________________                                        % C,       66.43;       % H,    9.92                                                     66.55                9.95                                          ______________________________________                                    

[α]_(D) ²⁵ -5.3° (CHCl₃, 1%) +3.6° (CH₃ OH, 1%).

The pooled fractions 161-182 contained a mixture of antibiotics X-14873Gand -H.

EXAMPLE 7 Preparation of the Thallium Salt of Antibiotic X-14873A

A solution of Antibiotic X-14873A-Na salt in methylene chloride waswashed with 1 N HCl, followed by water wash, then four times with anaqueous solution of thallium hydroxide. The solvent phase wasconcentrated, and by the addition of n-hexane crystalline thallium saltof Antibiotic X-14873A was recovered. Mp. 154° C.

Microanalysis: calcd for C₃₅ H₆₁ O₁₁ Tl (862.25): %C, 48.76; %H, 7.13;%Tl, 23.7; Found: %C, 49.01; %H, 7.07; %Tl, 21.91.

The structure of Antibiotic X-14873A was determined by X-ray analysis ofthe thallium salt.

EXAMPLE 8 Preparation of the Calcium Salt of Antibiotic X-14873A

A solution of Antibiotic X-14873A-Na salt in methylene chloride waswashed with 1 N HCl, followed by aqueous Ca(OH)₂ and water wash. Solventphase was concentrated in reduced pressure and was crystallized by theaddition of n-hexane. Mp. 133° C.

Microanalysis: Calcd for (C₃₅ H₆₁ O₁₁)₂ Ca (1355.84): %C, 62.01; %H,9.07; %Ca, 2.96; Found: %C, 62.17; H%, 9.28; %Ca, 2.96; [α]_(D) ²⁵ 3.5°(MeOH, 1%), 9.99° (CHCl₃, 1%).

Antibiotic X-14873A exhibits an oral toxicity (LD₅₀) in mice of 120mg/kg (24 hours); X-14873G exhibits an oral toxicity (LD₅₀) in mice of4000 mg/kg (24 hours); and X-14873H exhibits an oral toxicity (LD₅₀) inmice of 4000 mg/kg (24 hours).

Antibiotics exhibit some, albeit limited, activity against a variety ofbacteria and dermatophytes as indicated in the tables below.

    ______________________________________                                                        Minimum Inhibitory Concentration                                              (mcg/ml)                                                      Name of Organism                                                                              X-14873A                                                      ______________________________________                                        Staphylococcus aureus Smith                                                                    64                                                           Escherichia coli 257                                                                          128                                                           Salmonella typhi P58A                                                                         128                                                           Klebsiella pheumoniae A                                                                       128                                                           Proteus mirabilis 190                                                                         128                                                           Pseudomonas aeruginosa B                                                                      128                                                           Serratia marcescens SM                                                                        128                                                           Enterobacter cloacae 5699                                                                     128                                                           Trichophyton mentagrophytes                                                                   100                                                           Microsporum audouini                                                                          100                                                           ______________________________________                                    

    __________________________________________________________________________                ATCC Minimum Inhibitory Concentration (mcg/ml)                    Name of Organism                                                                          Number                                                                             X-14873A                                                                             X-14873H                                                                              X-14873G                                      __________________________________________________________________________    Streptococcus faecium                                                                     8043 0.1    2       63                                            Staphylococcus aureus                                                                     6538P                                                                              6.3    8       >1000                                         Sarcina lutea                                                                             9341 3.0    16      >1000                                         Bacillus megaterium                                                                       8011 3.0    4       >1000                                         Bacillus sp. E                                                                            27359                                                                              0.8    2       8                                             Bacillus subtilis                                                                         558* 3.0    16      >1000                                         Bacillus sp. TA                                                                           27860                                                                              3.0    8       >1000                                         Mycobacterium phlei                                                                       355  3.0    >1000   >1000                                         Streptomyces cellulosae                                                                   3313 12.5   >1000   >1000                                         Paecilomyces varioti                                                                      25820                                                                              125    >1000   >1000                                         Penicillum digitatum                                                                      26821                                                                              500    >1000   >1000                                         Candida albicans                                                                          477* 62.5   >1000   >1000                                         Saccharomyces cerevisiae                                                                  4226 500    >1000   >1000                                         __________________________________________________________________________     *NRRL number                                                             

As indicated above, antibiotics A, G and H possess the property ofadversely affecting the growth of certain gram-positive bacteria. Theyare, therefore, useful in wash solutions for sanitary purposes, as inthe washing of hands and the cleaning of equipment, floors orfurnishings of contaminated rooms or laboratories.

Antibiotic X-14873A has also been found to exhibit activity as a growthpromotant in ruminants, i.e., animals with a rumen function-for example,cattle. A discussion of the mechanism whereby feed is digested, degradedand metabolized in a ruminant animal can be found in U.S. Pat. No.3,839,557, issued Oct. 1, 1974, which discloses the use of certainantibiotics in improving ruminant feed utilization and is incorporatedherewith by reference. Economically important ruminant animals includecattle, sheep and goats.

The effectiveness of Antibiotic X-14873A in modifying the ratio ofvolatile fatty acids produced in the rumen (and thereby improve ruminantfeed utilization) is demonstrated by means of the in vitro testing.

Rumen fluid is obtained from a steer with a fistulated rumen. The steeris maintained on the following ration:

Corn: 89.93%

Alfalfa meal: 5.000%

Soy bean oil meal: 3.00%

Limestone: 0.80%

NaCl: 0.60%

Dicalcium phosphate: 0.50%

Trace minerals: 0.025%

Vitamin premix additions: 0.1%

Vitamin A, TIU: 4.0003

Vitamin D₃, IU: 0.801

Vitamin E, TIU: 3.002

The rumen fluid is immediately strained through a #30 mesh sieve. Foreach fermentation, 75 ml of the resulting fluid is added to a 250 mlflask containing the following:

1 g of 80%:20% finely ground grain: hay ration;

1 ml of an 18% aqueous glucose solution (1 millimole per flask);

1.5 ml of a 3.1% aqueous urea solution (0.76 millimole per flask);

60 micromoles of each of the 10 essential amino acids (arginine,histidine, leucine, methionine, threonine, valine, lysine, isoleucine,phenylalanine, tryptophan);

1 ml of an aqueous solution of test drug to give either 10 or 25 ug/ml(calculated total volume of fermentation mixture of 80 ml);

Each flask is incubated at 38° C. in a shaking water bath equipped witha gassing hood. Carbon dioxide is continuously passed through the hood.After four hours incubation, a 10 ml quantity of the fermentation fluidis centrifuged at 14,000 rpm (approximately 30,000 xg) for 20 minutes inan International Centrifuge equipped with a No. 874 angle head. Three mlof the supernate is added to 1 ml of a 25% metasphosphoric acid solutioncontaining 23 micromoles 2-methyl valeric acid as an internal standard.The resulting fluid is permitted to sit at room temperature for 30minutes. The fluid is filtered through a 0.22 millimicron Milliporefilter and refrigerated until gas-liquid chromatographic analyses forvolatile fatty acids.

Gas-liquid chromatographic (GLC) analyses of one nonmedicated controlfermentation and two fermentations, one with Antibiotic X-14873A and theother lasalocid, are set forth in the following table. The antibioticsare included in the rations at a concentration of 30 g/ton.

    ______________________________________                                                 VFA Ratio:.sup.(a) C.sub.3 /(C.sub.2 + nC.sub.4)                              Experimental Groups                                                  Experiment             Lasalocid,.sup.(c)                                                                       X-14873A.sup.(c)                            Day        Nonmedicated.sup.(b)                                                                      30 g/ton   30 g/ton                                    ______________________________________                                        Pre-medication                                                                1          0.262 ± 0.065                                                                          0.229 ± 0.013                                                                         0.213 ± 0.016                            3          0.260 ± 0.050                                                                          0.255 ± 0.051                                                                         0.235 ± 0.036                            5          0.240 ± 0.024                                                                          0.244 ± 0.060                                                                         0.234 ± 0.042                            8          0.240 ± 0.042                                                                          0.236 ± 0.041                                                                         0.245 ± 0.046                            10         0.206 ± 0.037                                                                          0.224 ± 0.011                                                                         0.216 ± 0.038                            Medication                                                                    2          0.211 ± 0.019                                                                          0.376 ± 0.109                                                                         0.445 ± 0.075                            5          0.248 ± 0.058                                                                          0.390 ± 0.073                                                                         0.584 ± 0.328                            7          0.325 ± 0.166                                                                          0.373 ± 0.015                                                                         0.679 ± 0.448                            9          0.338 ± 0.164                                                                          0.381 ± 0.064                                                                         0.642 ± 0.387                            12         0.293 ± 0.148                                                                          0.372 ± 0.056                                                                         0.495 ± 0.242                            ______________________________________                                         .sup.(a) ± one standard deviation.                                         .sup.(b) 4 animals.                                                           .sup.(c) 3 animals.                                                      

As shown in the above Table, the ratio of propionate (C₃) to acetate(C₂) and n-butyrate (nC₄) is significantly improved. With the increaseof propionates rather than acetates from the carbohydrates, theefficiency of carbohydrate and therefore feed utilization is increased.Also, it is shown that Antibiotic X-14873A elicited a greater increaseover control of the volitile fatty acid ratio than lasalocid, an activecompound.

Administration of Antibiotic X-14873A, hereafter "antibiotic" or"antibiotic compound" prevents and treats ketosis as well as improvesfeed utilization. The causative mechanism of ketosis is a deficientproduction of propionate compounds. A presently recommended treatment isadministration of propionic acid or feeds which preferentially producepropionates. It is obvious that encouraging propionate production fromordinary feeds will reduce incidence of ketosis.

It has been found that Antibiotic X-14873A increases the efficiency offeed utilization in ruminant animals when it is administered orally tothe animals. The easiest way to administer the antibiotic is by mixingit in the animal's feed.

However, the antibiotic can be usefully administered in other ways. Forexample, it can be incorporated into tablets, drenches, boluses, orcapsules, and dosed to the animals. Formulations of the antibioticcompound in such dosage forms can be accomplished by means of methodswell-known in the veterinary pharmaceutical art.

Capsules are readily produced by filling gelatin capsules with anydesired form of the desired antibiotics. If desired, the antibiotic canbe diluted with an inert powdered diluent, such as a sugar, starch, orpurified crystalline cellulose in order to increase its volume forconvenience in filling capsules.

Tablets of the antibiotic are made by conventional pharmaceuticalprocesses. Manufacture of tablets is a well-known and highly advancedart. In addition to the active ingredient, a tablet usually contains abase, a disintegrator, an absorbent, a binder, and a lubricant. Typicalbases include lactose, fine icing sugar, sodium chloride, starch andmannitol. Starch is also a good disintegrator as is alginic acid.Surface-active agents such as sodium lauryl sulfate and dioctyl sodiumsulphosuccinate are also sometimes used. Commonly used absorbents againinclude starch and lactose, while magnesium carbonate is also useful foroily substances. Frequently used binders are gelatin, gums, starch,dextrin and various cellulose derivatives. Among the commonly usedlubricants are magnesium stearate, talc, paraffin wax, various metallicsoaps and polyethylene glycol.

The administration of the antibiotic compound may be as a slow-pay-outbolus. Such boluses are made as tablets except that a means to delay thedissolution of the antibiotic is provided. Boluses are made to releasefor lengthy periods. The slow dissolution is assisted by choosing ahighly water-insoluble form of the antibiotic. A substance such as ironfiling is added to raise the density of the bolus and keep it static onthe bottom of the rumen.

Dissolution of the antibiotic is delayed by use of a matrix of insolublematerials in which the drug is inbedded. For example, substances such asvegetable waxes, purified mineral waxes, and water-insoluble polymericmaterials are useful.

Drenches of the antibiotic are prepared most easily by choosing awater-soluble form of the antibiotic. If an insoluble form is desiredfor some reason, a suspension may be made. Alternatively, a drench maybe formulated as a solution in a physiologically acceptable solvent suchas a polyethylene glycol.

Suspensions of insoluble forms of the antibiotic can be prepared innonsolvents such as vegetable oils, e.g., peanut, corn or sesame oil; ina glycol such as propylene glycol or a polyethylene glycol; or in water,depending on the form of the antibiotic chosen.

Suitable physiologically acceptable adjuvants are necessary in order tokeep the antibiotic suspended. The adjuvants can be chosen from amongthe thickeners, such as carboxymethylcellulose, polyvinylpyrrolidone,gelatin, and the alginates. Many classes of surfactants serve to suspendthe antibiotic. For example, lecithin, alkylphenol polyethylene oxideadducts, naphthalenesulfonates, alkylbenzenesulfonates, and thepolyoxyethylene sorbitan esters are useful for making suspensions inliquid nonsolvents.

In addition, many substances which effect the hydrophilicity, density,and surface tension of the liquid can assist in making suspensions inindividual cases. For example, silicone anti-foams, glycols, sorbitol,and sugars can be useful suspending agents.

The suspendable antibiotic may be offered to the grower as a suspension,or as a dry mixture of the antibiotic and adjuvants to be diluted beforeuse.

The antibiotic may also be administered in the drinking water of theruminants. Incorporation into drinking water is performed by adding awater-soluble or water-suspendable form of the antibiotic to the waterin the proper amount. Formulation of the antibiotic for addition todrinking water follows the same principles as formulation of drenches.

The most practical way to treat animals with the antibiotic compound isby the formulation of the compound into the feed supply. Any type offeed may be medicated with the antibiotic compounds, including commondry feeds, liquid feeds and pelleted feeds.

The methods of formulating drugs into animal feeds are well-known. It isusual to make a concentrated drug premix as a raw material for medicatedfeeds. For example, typical drug premixes may contain from about one toabout 400 grams of drug per pound of premix. The wide range results fromthe wide range of concentration of drug which may be desired in thefinal feed. Premixes may be either liquid or solid.

The formulation of ruminant feeds containing the proper amounts ofantibiotic for useful treatment is well understood. It is necessary onlyto calculate the amount of compound which it is desired to administer toeach animal, to take into account the amount of feed per day which theanimal eats and the concentration of antibiotic compound in the premixto be used, and calculate the proper concentration of antibioticcompound or of premix, in the feed.

All of the methods of formulating, mixing and pelleting feeds which arenormally used in the ruminant feed art are entirely appropriate formanufacturing feeds containing the antibiotic compound.

As has been shown, oral administration of the antibiotic beneficiallyalters the production of propionates relative to the production ofacetates in the rumen. It may therefore be postulated that the sametreatment would also benefit monogastric animals which ferment fibrousvegetable matter in the cecum, since it would be expected that abeneficial change in the propionate/acetate ratio would occur upon oraladministration of the instant antibiotic. Horses, swine and rabbits areexemplary animals which digest a part of their food by cecalfermentation.

Antibiotic X-14873A also has demonstrated activity as an agent in thetreatment or prevention of swine dysentery. The compound was examinedfor activity against Treponema hyodysenteriae, the etiologic agent ofswine dysentery. The compound exhibited a minimum inhibitoryconcentration of 5 mcg/ml.

Antibiotic X-14873 also exhibits activity as a coccidio-static agent,exhibiting in vitro activity against E. tenella at 200 ppm.

What is claimed:
 1. A compound of the formula ##STR9## and thepharmaceutically acceptable salts thereof.
 2. A compound of the formula##STR10##
 3. A compound of the formula ##STR11##